SHH E176/E177-Zn2+ conformation is required for signaling at endogenous sites

Diana S. Himmelstein; Ivelisse Cajigas; Chunming Bi; Brian S. Clark; Grant Van Der Voort; Jhumku D. Kohtz

doi:10.1016/j.ydbio.2017.02.006

SHH E176/E177-Zn²⁺ conformation is required for signaling at endogenous sites

Diana S. Himmelstein, Ivelisse Cajigas, Chunming Bi, Brian S. Clark, Grant Van Der Voort, Jhumku D. Kohtz^*

^*Corresponding author for this work

Neurology

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

Abstract

Sonic hedgehog (SHH) is a master developmental regulator. In 1995, the SHH crystal structure predicted that SHH-E176 (human)/E177 (mouse) regulates signaling through a Zn²⁺-dependent mechanism. While Zn²⁺ is known to be required for SHH protein stability, a regulatory role for SHH-E176 or Zn²⁺ has not been described. Here, we show that SHH-E176/177 modulates Zn²⁺-dependent cross-linking in vitro and is required for endogenous signaling, in vivo. While ectopically expressed SHH-E176A is highly active, mice expressing SHH-E177A at endogenous sites (Shh^E177A/-) are morphologically indistinguishable from mice lacking SHH (Shh^-/-), with patterning defects in both embryonic spinal cord and forebrain. SHH-E177A distribution along the embryonic spinal cord ventricle is unaltered, suggesting that E177 does not control long-range transport. While SHH-E177A association with cilia basal bodies increases in embryonic ventral spinal cord, diffusely distributed SHH-E177A is not detected. Together, these results reveal a novel role for E177-Zn²⁺ in regulating SHH signaling that may involve critical, cilia basal-body localized changes in cross-linking and/or conformation.

Original language	English (US)
Pages (from-to)	221-235
Number of pages	15
Journal	Developmental Biology
Volume	424
Issue number	2
DOIs	https://doi.org/10.1016/j.ydbio.2017.02.006
State	Published - Apr 15 2017

Funding

We thank E.A. Garber (Biogen) for the human Shh-E176A and ShhN-E176A cDNA constructs, D. Baker (Biogen) for uSHHN protein, A.McMahon (Harvard) for ShhN/+ mice, T. Caspary (Emory) for Wim mice, D.S.H.'s thesis committee (J. Kessler, A. Chenn, and R. Miller, Northwestern University) for helpful comments throughout the course of this work, N. Copeland (National Cancer Institute) for EL250 cells and recombineering plasmids PL253 and PL452, D. Epstein (U Penn) for Shh in situ probe, J. Rubenstein (UCSF) for Dlx2 in situ probe, and the following lab members for technical expertise and advice: M. VanGompel (recombineering and plasmid design), J.C. Savage (initial characterization of α-SHHCL/P), and J. Feng (early charactrerization of SHH-E176A and SHHN-E176A proteins, including the Western in Fig. S1). Antibodies shown in Fig. 5M-a and E1 anti-SHH were obtained from the Developmental Studies Hybridoma Bank. This work is funded by NIMH R01MH094653 to J.D.K. The manuscript has been approved by all authors.

ASJC Scopus subject areas

Molecular Biology
Developmental Biology
Cell Biology

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10.1016/j.ydbio.2017.02.006

Cite this

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title = "SHH E176/E177-Zn2+ conformation is required for signaling at endogenous sites",

abstract = "Sonic hedgehog (SHH) is a master developmental regulator. In 1995, the SHH crystal structure predicted that SHH-E176 (human)/E177 (mouse) regulates signaling through a Zn2+-dependent mechanism. While Zn2+ is known to be required for SHH protein stability, a regulatory role for SHH-E176 or Zn2+ has not been described. Here, we show that SHH-E176/177 modulates Zn2+-dependent cross-linking in vitro and is required for endogenous signaling, in vivo. While ectopically expressed SHH-E176A is highly active, mice expressing SHH-E177A at endogenous sites (ShhE177A/-) are morphologically indistinguishable from mice lacking SHH (Shh-/-), with patterning defects in both embryonic spinal cord and forebrain. SHH-E177A distribution along the embryonic spinal cord ventricle is unaltered, suggesting that E177 does not control long-range transport. While SHH-E177A association with cilia basal bodies increases in embryonic ventral spinal cord, diffusely distributed SHH-E177A is not detected. Together, these results reveal a novel role for E177-Zn2+ in regulating SHH signaling that may involve critical, cilia basal-body localized changes in cross-linking and/or conformation.",

author = "Himmelstein, {Diana S.} and Ivelisse Cajigas and Chunming Bi and Clark, {Brian S.} and {Van Der Voort}, Grant and Kohtz, {Jhumku D.}",

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AU - Himmelstein, Diana S.

AU - Cajigas, Ivelisse

AU - Bi, Chunming

AU - Clark, Brian S.

AU - Van Der Voort, Grant

AU - Kohtz, Jhumku D.

PY - 2017/4/15

Y1 - 2017/4/15

N2 - Sonic hedgehog (SHH) is a master developmental regulator. In 1995, the SHH crystal structure predicted that SHH-E176 (human)/E177 (mouse) regulates signaling through a Zn2+-dependent mechanism. While Zn2+ is known to be required for SHH protein stability, a regulatory role for SHH-E176 or Zn2+ has not been described. Here, we show that SHH-E176/177 modulates Zn2+-dependent cross-linking in vitro and is required for endogenous signaling, in vivo. While ectopically expressed SHH-E176A is highly active, mice expressing SHH-E177A at endogenous sites (ShhE177A/-) are morphologically indistinguishable from mice lacking SHH (Shh-/-), with patterning defects in both embryonic spinal cord and forebrain. SHH-E177A distribution along the embryonic spinal cord ventricle is unaltered, suggesting that E177 does not control long-range transport. While SHH-E177A association with cilia basal bodies increases in embryonic ventral spinal cord, diffusely distributed SHH-E177A is not detected. Together, these results reveal a novel role for E177-Zn2+ in regulating SHH signaling that may involve critical, cilia basal-body localized changes in cross-linking and/or conformation.

AB - Sonic hedgehog (SHH) is a master developmental regulator. In 1995, the SHH crystal structure predicted that SHH-E176 (human)/E177 (mouse) regulates signaling through a Zn2+-dependent mechanism. While Zn2+ is known to be required for SHH protein stability, a regulatory role for SHH-E176 or Zn2+ has not been described. Here, we show that SHH-E176/177 modulates Zn2+-dependent cross-linking in vitro and is required for endogenous signaling, in vivo. While ectopically expressed SHH-E176A is highly active, mice expressing SHH-E177A at endogenous sites (ShhE177A/-) are morphologically indistinguishable from mice lacking SHH (Shh-/-), with patterning defects in both embryonic spinal cord and forebrain. SHH-E177A distribution along the embryonic spinal cord ventricle is unaltered, suggesting that E177 does not control long-range transport. While SHH-E177A association with cilia basal bodies increases in embryonic ventral spinal cord, diffusely distributed SHH-E177A is not detected. Together, these results reveal a novel role for E177-Zn2+ in regulating SHH signaling that may involve critical, cilia basal-body localized changes in cross-linking and/or conformation.

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